ATP-diphosphohydrolases, process of purification thereof and process of producing thereof by recombinant technology

ABSTRACT

The present invention relates to two ATP diphosphohydrolases (ATPDase enzymes) isolated from bovine aorta and pig pancreas, which enzymes have a molecular weight for their catalytic unit of about 78 and 54 Kilodaltons, respectively. A first process for obtaining a highly purified ATPDase is also an object of the present invention. This process has been successfully applied to the purification of both the pancreatic and the aorta enzymes and is deemed to work in the purification of any ATPDase. For both sources of enzymes, the process allows the specific activity of the enzyme to be increased by at least 10,000 fold when compared to the activity retrieved in the crude cell homogenates. The novel process involves an ion exchange chromatography step, a separation on an affinity column, followed by an electrophoresis under non-denaturing conditions. The two enzymes purified by this process (aortic and pancreatic) are glycosylated and, when deglycosylated, have molecular weights shifted to about 56 and 35 Kdaltons, respectively. Partial amino acid sequences have been obtained for each enzyme. The partial sequences appear highly homologous with a human lymphoid cell activation antigen named CD39. An antibody directed against the porcine pancreatic enzyme cross-reacts with a protein present in endothelial cell lines and in bovine aorta (78 KDa). The high degree of homology of the pancreatic and aortic enzymes with CD39 and their cross-reactivity are indications that both enzymes are related. The pancreatic enzyme completely lacks the first 200 amino acids of CD39, which means the ATPDase activity is comprised between residues  200  and  510  of CD39. Since this is the first time that a sequence is assigned to ATPDases, a second new process for producing ATPDases by recombinant technology can also be used. Therefore a second new process for producing an ATPDase using the CD39-encoding nucleic acid or part or variant thereof is also described.

FIELD OF THE INVENTION

[0001] The present invention relates to a process of purification tohomogeneity of ATP-diphosphohydrolases involved in numerous nucleotideand nucleoside receptor-mediated physiological functions, namelyplatelet aggregation, vascular tone, secretory, inflammatory andexcretory functions and neurotransmission. These enzymes, which havebeen particularly obtained from bovine aorta and pig pancreas have beenpurified and their catalytic unit identified. The partial amino acidsequences of each ATPDase show a high degree of homology with a lymphoidcell activation system named CD39.

BACKGROUND OF THE INVENTION

[0002] ATP-diphosphohydrolases (ATPDases) or apyrases (EC 3.6.1.5) havebeen found in plants, invertebrates and vertebrates. The enzymecatalyses the sequential hydrolysis of the ν- and β-phosphate residuesof triphospho- and diphosphonucleosides. These enzymes are generallyactivated in the presence of divalent cations Ca⁺² or Mg⁺² and inhibitedby sodium azide. In plants, the enzymes are found in the cytoplasm, insoluble or membrane-associated forms, and are generally more active atacid pH. Their precise function is not known, but there is some evidencethat they are involved in the synthesis of carbohydrates. Ininvertebrates, the enzymes are more active at neutral or alkaline pH.Found mainly in saliva and in salivary glands of hematophagous insects,an antihemostatic role has been demonstrated. In vertebrates, a limitednumber of studies have already defined a diversity of ATPDases. Thecatalytic site of these enzymes is generally exposed to extracytoplasmicspaces (ectoenzymes). By their location and kinetic properties, thesedifferent types of ATPDases could influence the main systems of theorganism, namely vascular and nervous systems. Their specific role inthese systems is determined by the presence of purine and pyrimidinereceptors which react with triphosphonucleosides and their derivativesat the surface of numerous cell types.

[0003] Presence of both ectoATPase and ectoADPase activities in thevascular system has been known for many years, and up until the work ofYagi et al. (1989), they were attributed to two distinct enzymes. Thelatter purified these activities and showed that in bovine aorta, asingle enzyme was responsible for the sequential hydrolysis of ATP andADP. A mammalian ATPDase had been first described in the pancreas (Lebelet al., 1980) and was further reported in several other tissues. Yagi etal. (1989) proposed that the enzyme from aorta was similar to thepreviously reported mammalian ATPDase from pancreas and that it wasassociated with the intima of bovine aorta. Purification to homogeneitywas demonstrated by SDS-polyacrylamide gel electrophoresis (PAGE) andsilver staining. The apparent molecular weight of the pure enzyme wasestimated at 110 KDa. The existence of the ATPDase in the bovine aortawas corroborated by Côté et al. (1991) who, by showing that identicalheat and irradiation-inactivation curves with ATP and ADP as substrates,assigned to the same catalytic site the ATPase and ADPase activities. Acomparison of the biochemical properties led C{circumflex over (0)}té etal. supra to propose that the bovine aorta enzyme was different from thepancreas ATPDase. Indeed, the enzymes have different native molecularweights, optimum pH and sensitivities to inhibitors. They proposed toidentify pancreas enzyme as type I and the aorta enzyme as type II. Inthe bovine aorta, the enzyme was found to be associated with smoothmuscle cells and endothelial cells and could inhibit ADP-inducedplatelet aggregation. Côté et al. (1991) further showed that concurrentaddition of ATPDase and ATP to platelet-rich plasma resulted in animmediate dose-dependent platelet aggregation caused by the accumulationof ADP, followed by a slow desaggregation attributable to its hydrolysisto AMP. In the absence of ATPDase, ATP did not induce any aggregationwhile ADP initiate an irreversible aggregation which extent is limitedby the ADPase activity of the enzyme. ATPDase also attenuated theaggregation elicited by thrombin and collagen but not by PAF (PlateletActivating Factor), the first two agonists having an effect mediated byplatelet ADP release. It was therefore suggested that ATPDase had a dualrole in regulating platelet activation. By converting ATP released fromdamaged vessel cells into ADP, the enzyme induced platelet aggregationat the sites of vascular injury. By converting ADP released fromaggregated platelets and/or from hemolyzed red blood cells to AMP, theATPDase could inhibit or reverse platelet activation, and consequentlylimit the growth of platelet thrombus at the site of injury. In theirattempt to further characterize the aorta ATPDase, the present inventorshave developed a new process for producing highly purified ATPDases.They have established a procedure by which its specific activity can beincreased over and above the activity of a crude cell preparation bymore than 10000-fold. They also discover that the purified enzyme (thecatalytic unit) had a molecular weight different from the one previouslyreported for the native form of the enzyme (190 KD by using theirradiation technique), suggesting that the enzyme may exist in amultimeric form in its native state. Partial amino acid sequences ofboth bovine aorta and porcine pancreatic ATPases have been obtained.

[0004] In a completely different field, Maliszenski et al. (1994) havepublished the sequence of a human lymphoid cell activation antigendesignated CD39. Another group (Christoforidis et al. 1995) describedthe purification of a human placenta ATPDase of a molecular weight of 82KDa. Its partial amino acid sequence shows a high degree of homologywith CD39.

[0005] When the above mentioned partial amino acid sequences wereentered in GenBank for verifying the presence of any homologoussequence, complete homology was surprisingly found for some of thesefragments with the CD39 gene product. The complete sequences of theATPDases remain to be obtained. Assuming that CD39 is an up to dateunknown ATPDase, a process for producing ATPDases by recombinanttechnology is now possible, and CD39 can now be used to reduce plateletaggregation and thrombogenicity.

STATEMENT OF THE INVENTION

[0006] It is an object of the present invention to provde two ATPDasesisolated from bovine aorta and porcine pancreas, which enzymes have amolecular weight for their catalytic unit of about 78 and 54Kilodaltons, respectively. A novel process for obtaining a highlypurified ATPDase is also an object of the present invention. Thisprocess has been successfully applied to the purification of both thepancreatic and the aorta enzymes and is deemed to work in thepurification of any ATPDase. For both sources of enzymes, the processallows the specific activity of the enzyme to be increased by at least300 fold when compared to the activity retrieved in the microsomialfraction of these cells as previously reported for an aortic andpancreatic proteins of a native molecular weight of about 190 and 130KDa, respectively.

[0007] The two ATPDases purified to homogeneity were partiallysequenced. These sequences have shown striking similarities with a humanlymphoid cell activation antigen named CD39 (Maliszenski et al., 1994).Since the molecular weight of CD39 and its glycosylation rate appears todefine a human counterpart for the present bovine aortic ATPDase, it isthe first time that a sequence is assigned to an ATPDase. A process ofproducing an ATPDase by recombinant technology is now possible using ahost cell expressing the CD39 human protein, its homologous sequences inbovine and porcine species, and variants and parts thereof.

[0008] The present invention also relates to the use of CD39 and of theabove bovine and porcine homologous proteins for reducing plateletaggregation and thrombogenicity.

DESCRIPTION OF THE PRESENT INVENTION

[0009] The research team to which the present inventors belong hasalready characterized the pig pancreatic ATPDase, and the latterreassessed the properties of the bovine aorta enzyme. They confirmedthat the aorta ATPDase was different from its pancreatic counterpart.They have found previously (Côté et al., 1992) that the aorta enzyme(isolated from a microsomal fraction of the cells) had a molecularweight of about 190 kDa in its native state. In their work forextensively purify this enzyme, they found that the highly purifiedenzyme had a molecular weight on SDS-PAGE of about 78 KDa. Yagi et al.(1989) have already shown that an ATPDase purified to homogeneity had amolecular weight of 110 KDa. After purifying the enzyme by the presentmethod, the 110 kDa band was indeed absent from SDS-PAGE. A unique bandmigrating of an estimated weight of 78 KDa was rather revealed. Theconfirmation of the identity of the purified enzyme was achieved bybinding FSBA, an ATP analog binding the enzyme, to the separated andblotted enzyme. The use of anti-FSBA antibodies revealed the presence ofthe bound enzyme and this binding was inhibited with ATP and ADP. Thesame procedure was applied to confirm the identification of the pancreasATPDase Type I.

[0010] The present process allows the purification of ATPDases to a veryhigh level. In the aorta, the purified enzyme has a specific activitywhich is increased by at least 300 fold compared with the specificactivity of microsomal fraction (already enriched by about 30 fold fromthe crude cell preparation).

[0011] The bovine aorta and porcine pancreatic ATPDases have beenpartially sequenced, and the sequences have been found to be highlyhomologous to a human lymphoid cell activation antigen designated CD39(Maliszenski et al.,op. cit.). The complete sequences of the ATPDasestypes I and II have not been obtained yet. If one assumes that CD39 geneproduct is an ATPDase type II, the present invention thereforecontemplates the use of CD39 in the reduction of platelet aggregationand of thrombogenicity, as well as a process of making ATPDases usingthe CD39 sequence, variants or parts thereof (recombinant technology).

[0012] The present invention will be described hereinbelow withreference to the following Examples and Figures which purpose is toillustrate rather than to limit the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 illustrates the protein composition of the bovine aortaATPDase (type II) at the different purification steps as determined bySDS-PAGE. Electrophoresis was run in a 7-12.5% polyacrylamide gel.Proteins were stained with Coomassie Blue or silver nitrate dye. MWstandards: 97.4, 66.2, 45.0, 31.0, 21.5, 14.4 KDa; particulate fraction(part. fract.), 100 μg; DEAE-agarose fraction, 35 μg; Affi-Gel bluefraction, 20 μg; the lower band of activity was cut out from thenon-denaturing gel (N.D. gel); sample buffer alone (Control).

[0014]FIG. 2 illustrates a Western blot of FBSA labelled protein(ATPDase type II) isolated from Affi-Gel blue column. Labelled proteinswere separated on a 8-13.5% gradient gel by SDS-PAGE, transferred toImmobilon-P membrane, incubated with a rabbit antibody anti-FBSA(1:10,000) and detected by a secondary antibody conjugated to alkalinephosphatase (1:6,000). Twenty μg of protein from Affi-Gel blue columnfraction was used for the assays: incubation with FBSA (FBSA);incubation with FBSA with competing Ca-ATP (FBSA+ATP); incubationwithout FBSA (no FBSA). MW standards are the same as in FIG. 1.

[0015]FIG. 3 illustrates the SDS-PAGE protein patterns at the differentsteps of the purification procedure and after N-glycosidase F digestionof the Affi-Gel blue fraction. Protein samples were fractionated on a8-13.5% polyacrylamide gradient. A) One unit of N-glycosidase F (silvernitrate stain); B) Six μg from the Affi-Gel blue fraction incubated for12 h without N-glycosidase F (silver nitrate stain); C) Idem as B with 1unit of N-glycosidase F (silver nitrate stain); A′) Same as A (Coomassieblue stain); B′) Same as B (Coomassie blue stain); C′) Same as C(Coomassie blue stain); D) MW standards: 97.4, 66.2, 45.0, 31.0, 21.5,14.4 kDa (Coomassie blue stain), E) ZGM (zymogen granule membrane), 60μg (Coomassie blue stain); F) Active fraction from DEAE-agarose column,25 μg (Coomassie blue stain); G) Active fraction from Affi-Gel bluecolumn, 6 μg (Coomassie blue stain); G′) Same as G (silver nitrateoverstain); H) Activity band located after PAGE under non-denaturingconditions (silver nitrate overstain); I) Control, band located justabove the activity band after PAGE under non-denaturing conditions(silver nitrate overstain).

[0016]FIG. 4 shows a Western blot of FSBA labelled samples of thepancreatic enzyme type I fraction. Labelled sample were loaded on a7-12% polyacrylamide SDS-gel, transferred to Immobilon-P membrane,incubated with the rabbit antibody anti-FSBA and detected by a secondaryantibody conjugated to alkaline phosphatase. Six μg of Affi-Gel bluecolumn were used in lanes B), C) and D). A) MW standards: 97.4, 66.2,45.0, 31.0, 21.5, 14.4 kDa; B) FSBA; C) FSBA+competing ADP; D) Nolabelling.

[0017]FIG. 5 shows a Western blot of human endothelial cell extractslabelled with an antibody directed against a fragment common to ATPDasetype I and CD39. The ATPDase type II (78KDa) is clearly detected as wellas low amounts of ATPDase type I (54KDa).

EXAMPLE 1 Purification of the ATPDase Type II

[0018] a) Isolation of the Particulate (Microsomal) Fraction from theBovine Aorta

[0019] Bovine aorta, obtained from a local slaughterhouse, were kept onice and processed within one hour after the death of the animals. Allsteps were carried out at 4° C. The inner layer was stripped outmanually, passed through a meat grinder, and homogenized (10%) with aPolytron™ in the following solution: 95 mM NaCl, Soybean TrypsinInhibitor (20 μg/mL), 0.1 mM Phenyl-methyl-sulphonyl-fluoride (PMSF) and45 mM Tris-HCl pH 7.6. After filtering with cheesecloth, the homogenatewas centrifuged at 600 X g for 15 minutes with a Beckman JA-14centrifuge at 2100 RPM. The supernatant was recovered and centrifuged at22,000 X g for 90 minutes with the same centrifuge at 12,000 RPM. Theresulting pellet was suspended in 0.1 mM PMSF and 1 mM NaHCO₃ pH 10.0with a Potter Elvejehm™ homogenizer at a dilution of 3 to 6 mg ofprotein per mL. The suspension was loaded on a 40% sucrose cushion andcentrifuged at 100,000 X g for 140 minutes with a SW 28 Beckman rotor.The enzyme was recovered on the cushion and kept at 4° C. overnight.This membrane preparation was then suspended in 12 volumes of 0.1 mMPMSF and 1 mM NaHCO₃ pH 10.0 and centrifuged at 240,000 X g for 45minutes in a SW 50.2 Beckman rotor. The pellet was rinsed twice: oncewith o 0.1 mM PMSF and 30 mM Tris-HCl ph 8.0 and once with 2 mM EDTA and30 mM Tris-HCl pH 8.0. The final pellet was suspended in 7.5% glycerinand 5 mM Tris-HCl ph 8.0 at a concentration>1 mg of protein per mL andfrozen at −20° C., or directly solubilized. At this stage, the specificactivity of the ATPDase was enriched by about 33 fold.

[0020] b) Solubilization and Column Chromatographies

[0021] The particulate fraction (pf) was solubilized with 0.3% TritonX-100™ and 30 mM Tris-HCl pH 8.0 at a concentration of 1 mg/mL proteinand centrifuged at 100,000 X g for 1 hour in a SW 50.2 Beckman rotor.All further steps involving a detergent are practised with Triton X-100,but any similar detergent (a non-ionic detergent) may be used forachieving the purpose of this invention. The supernatant was loaded onan ion exchange column, preferably containing diethylaminoethyl (DEAE),like DEAE-Bio Gel A Agarose™, preequilibrated with 0.1% Triton X-100™,7.5% glycerin and 10 mM Tris-HCl pH 8.0. The protein was eluted in thesame buffer by a NaCl gradient (0.03 to 0.12 M), followed by a 0.1%Triton X-100™ and 2 M NaCl wash. Active fractions were pooled in 0.1Xbuffer E (5X buffer E: 0.5% Triton X-100™, 960 mM glycine, 125 mMTris-HCl pH 7.0) and electrodialysed in 15 mL cuvettes by an ISCO™electro-eluter according to the following technique: 1X buffer E wasloaded in the apparatus and a 15 mA current was applied per cuvette. The1X buffer E was changed 4 times at 50 minute intervals. The dialysatewas equilibrated at pH 5.9 with 200 mM histidine adjusted to pH 4.0 withHCl (about 20 mM final) and loaded on an Affi-Gel™ blue columnpreequilibrated with 0.07% Triton X-100™, 7.5% glycerin, 30 mM histidineand 30 mM Tris-HCl pH 5.9. Proteins were eluted by a linear gradientfrom 100% buffer A to 100% buffer B (buffer A (80 ml): 0.07% TritonX-100™, 7.5% glycerin and 10 mM Tris-HCl pH 6.5; buffer B (80 ml): 1MNaCl, 0.07% Triton X-100™, 7.5% glycerin and 10 mM Tris-HCl pH 7.5),followed by a 1M NaCl, 0.1% Triton X-100™, 100 mM Tris-HCl pH 8.5 wash.The active fraction was dialysed against 0.05% Triton X-100™, 1 mMTris-HCl pH 8.0, concentrated on a 1 ml DEAE-agarose column as describedabove, eluted in 0.4 M NaCl, 0.07% Triton X100™, 10 mM Tris-HCl pH 8.0and dialysed against distilled water.

[0022] c) Separation by Polyacrylamide Gel Electrophoresis (PAGE) underNon-denaturing Conditions

[0023] This type of gel allows for separating proteins upon theirmolecular weight and electrical charge while preserving their activityin such a way that this activity can be measured after migration. Twopolyacrylamide preparations were poured between two glass plates to forma gradient and polymerized. The 4% acrylamide solution was composed of:4.5 mL of separating buffer (Tris 1.5 M pH 8.8+0.4% Triton X-100™), 2.5mL acrylamide 30%, 180 μL Na deoxycholate 10%, water up to 18 mL, 60 μLAPS 10% and 7 μL TEMED. The 7.5% acrylamide solution was composed of thesame ingredients except for the volume of acrylamide: 4.5 mL. A stackinggel was extemporaneously prepared and poured at the top of theseparating gel, the stacking gel was composed of: 2.5 mL of stackingbuffer (Tris-base 0.5 M pH 6.8), 6.1 mL of water, 1.34 acrylamide 30%,0.1 mL Na deoxycholate 10%, 0.1 mL Triton X-100™, 50 μL APS 10% and 10μL TEMED. Wells are formed in this layer during polymerization. Twovolumes of the sample obtained after DEAE-agarose or Affigel Bluecolumns were added to one volume of sample buffer of the followingcomposition to obtain about 100 μg proteins: 0.07% (v/v) Triton X-100™,1.5% (w/v) Na deoxycholate, 10% glycerol, 65 mM Tris-base and 0.005%bromophenol blue. The suspended sample was allowed to stand 10 minuteson ice and centrifuged. The supernatant was loaded on gel. The proteinswere migrated at 4° C. at a 20 mAmp power in reservoir buffer (0.1%Triton X-100, 0.1% sodium deoxycholate, 192 mM glycine and 25 mM Tris pH8.3). For revealing activity in the separated bands, the latter wereplaced in a dosage buffer (Tris-base 66.7 mM, imidazole 66.7 mM, CaCl₂10 mM, pH 7.5). After preliminary incubation for 30 minutes at 37° C.,the substrate (ADP or ATP) 5 mM was added. After 2 to 10 minuteincubation, a white calcium phosphate precipitate significative of ATPdiphosphohydrolase activity is formed. Three bands are seen for theaorta enzyme and one for the pancreas (these bands were all revealed ongel by silver overstaining). For further characterization, the mostactive band was loaded on an SDS-PAGE according to Laemmli (1970) and asingle band appeared on the gel after silver nitrate staining, which isindicative of an enzyme purification to homogeneity after thenon-denaturing gel. FIG. 1 shows the high sensitivity of detectionconferred by the use of silver staining compared to a conventionalCoomassie blue staining (see lanes 4 and 5). The active band purifiedfrom the gel has a molecular weight of 78 KDa when migrated on SDS-PAGE.

[0024] d) ATPDase Assays during Chromatographic Steps

[0025] Enzyme activity was determined at 37° C. in the followingincubation medium: 50 mM Tris-imidazole (pH 7.5), 8 mM CaCl₂ and 0.2 mMsubstrate (ATP or ADP). Phosphorus was measured by the malachite greenmethod according to Baykov et al. (1988). One unit of enzyme correspondsto the liberation of 1 μmol of phosphate per minute per mg of protein at37° C. Proteins were estimated by the technique of Bradford (1976).

[0026] The ATPDase activity retrieved in isolated fractions aresummarized in the following Table: TABLE 1 ATPDase purification of thebovine aorta ATPDase type II Total Total Specific PurificationHydrolysis protein activity activity Yield factor rate Step mg unitsunits/mg % -fold ATP/ADP Particulate fraction (pf) 293 263 0.9 —  (33)*1.5 pf + Triton X-100 293 117 0.4 100  1 1.4 100,000 g supernatant of186 91.2 0.5 78   1.2 1.3 solubilized pf DEAE column 15.1 72.2 4.8 62  11.9 1.1 Affi-Gel blue column 2.76 57.8 21 49 53 1.1 Con A 0.61 33.555 29 138  1.1

[0027] e) Confirmation of the Identity of ATPDase

[0028] The fraction eluted from Affi-gel was labelled with 5′-p-fluorosulfonylbenzoyl adenosine (FBSA), a marker which formscovalent bonds with adenosine-binding proteins. FSBA blocked the enzymeactivity and excess of ATP or of ADP prevents this effect. In addition,FSBA efficiently bound the purified enzyme, as monitored by a Westernblot technique using an antibody directed to FSBA, which binding isprevented in the presence of ATP (see FIG. 2) or ADP (data not shown).

[0029] The results obtained on SDS-PAGE shows that the enzyme waspurified to homogeneity when using the successive steps ofsolubilization of the particulate fraction, first purification on an ionexchange column, second purification on an affinity column and thirdpurification on non-denaturing electrophoretic conditions. The AffigelBlue column did not achieve purification to homogeneity but allowed amuch higher recovery then the 5′ AMP-Sepharose™ used by Yagi et al.(about 7 fold higher). Moreover, the use of the Affigel column and thenon-denaturing gel allowed us to purify an enzyme that is different fromthe one disclosed by Yagi.

[0030] f) ATPDases are Glycosylated Proteins

[0031] Purification on Concanavalin A column

[0032] Further purification of the Affi-Gel blue fraction of aortaenzyme was also obtained with Con A agarose column. Briefly, Con A (4 mlbeads) and the protein sample from the Affi-Gel blue column werepreequilibrated with 0.05 % Triton X-100, 100 mM NaCl, 1 mM CaCl₂, 1 mMMnCl₂ and 20 mM PIPES, pH 6.8, at room temperature. The protein samplewas passed through the column at a flow rate of 3 ml/h, 40 ml of thepreequilibration buffer was then added to wash the unbound materials ata flow rate of 10 ml/h. The activity was eluted with 20 ml of 0.5 MMe-α-D-mannopyranoside diluted in the preequilibration buffer. Thepurified sample was dialysed and concentrated on a mini-DEAE column asdescribed above.

[0033] Precipitation of ATPDase Activity with Lectin-agarose

[0034] Four lectins conjugated to agarose were tried: Con A, WGA,Soybean agglutinin and UEA. Experiments were carried out at roomtemperature for Con A, and at 4° C. for the other agglutinins. Onehundred μl of each 50% slurry were put in a microcentrifuge tube andwashed 4 times with buffer A: 0.05 % Triton X-100, 100 mM NaCl and 20 mMPIPES pH 6.8. In the case of Con A, 1 mM CaCl₂ and 1 mM MnCl₂ were addedto this buffer. Twenty μg of ATPDase purified from the Affi-Gel bluecolumn, equilibrated in buffer A, were added to the lectin-agarose beadsand rocked for 45 min, then centrifuged for 1 min. The supernatant waskept and the beads were washed 3 times with 1 ml buffer A. Protein boundto the lectins was eluted with 150 μl of 500 mM of the appropriate sugarin buffer A, rocked for 30 min and centrifuged. The elution step wasrepeated once and the 2 eluates were pooled. The sugar used to eluateproteins from Con A, WGA, Soybean and UEA were Me-α-D-mannopyranoside,D-GlcNAc, D-GalNAc and L-Fuc respectively. TABLE 2 ATPase binding tolectins Lectin- Relative ADPase Presence of the 78 agarose Fractionsactivity kDa band on SDS-PAGE Sugar specificity Con A Supernatant  5%traces Mannose, Bound  95% Glucose Eluted  62% + WGA Supernatant  5%traces GlcNAc, NeuNAc, Bound  95% Mannose structure § Eluted  69% +Sialic acid § Soybean Supernatant 100% + GalNAc Bound  0% Eluted  0% −UEA Supernatant 100% + Fucose Bound  0% Eluted  0% − # fraction were puton SDS-PAGE, stained with silver nitrate, and looked for the presence ofthe 78 kDa. The sugar specificity of each agglutinin is also presented.

[0035] Only WGA bound the ATPDase type II as for Con A. ATPDase bindingto these two lectins is indicative of a specificity for the sugarsglucose and/or mannose and/or GlcNAc (Glucosamine-N-Acetyl) and/orNeuNAc (Neuraminic-N-Acetyl).

[0036] The deglycosylated form had a molecular weight of about 56 KDa,which suggests that about 5 to 11 glycosyl chains are present on the 78KDa protein (assuming that a glycosyl group may have a molecular weightof 2 to 4 KDa).

Example 2

[0037] Purification of the ATPDase Type I

[0038] The procedure described in Example 1 has been followed forpurifying the pancreatic ATPDase type I enzyme, starting from thezymogen granule membrane of pig pancreas.

[0039] In deglycosylation experiments, the molecular weight of thecatalytic unit has been shown to be shifted from 54 to 35 KDa.Therefore, the chemical procedure exemplified above is deemed to applyto the purification of ATPDases in general.

[0040] h) Level of Enrichment

[0041] The level of enrichment is determined from the data shown inTable 1 for aorta ATPDase type II and from the following Table 3obtained for pancreatic ATPDase type

[0042] From the crude cell preparation to the Affigel Blue column, theenzymes of both pancreatic and aorta sources were purified to at least a1600 fold level (see Tables 1 and 3. After the non-denaturing gel, thequantity of proteins falls under the detection level of the method used,which renders difficult the calculation of a specific activity. However,one can roughly estimate the process to reach about a 10 thousand foldpurification, as judged by the density of the ATPDase reaction bandrelative to other proteins on the non-denaturing electrophoretic gel.

[0043] Referring to Table 1, the lectin-binding step is not consideredproperly as an essential step of the purification process. This step hasbeen added to show that the aorta ATPDase is a glycoprotein which, whendeglycosylated, shifts from a molecular weight of 78 KDa to a molecularof 56 KDa (representing the proteic backbone). Since the lectin-bindingstep does not achieve the obtention of a pure protein, the mostconvenient way to obtain a pure protein is to submit the crude cellpreparation sequentially to the ion exchange chromatography, the AffigelBlue chromatography and to non-denaturing gel electrophoresis. Theidentity of the protein is then confirmed by ATP-labelling with FSBA.

Example 3

[0044] Partial Amino Acid Sequences

[0045] CNBr digests have been obtained from the purified bovine aortaand porcine pancreatic ATPDases. The sequences of the digests are asfollows: SEQ. ID. NO.: Bovine aorta ATPDase: Glu Thr Pro Val Tyr Leu GlyAla Thr Ala Gly 3                   5                  10 Leu Leu ArgMet Glu 4                   5 Ala Asp Lys Ile Leu Ala Asn Xaa Val Ala 5                  5                  10 Ser Ser Ile Tyr Pro Phe Asp PheGln Gly Ala Arg Ile 6                   5                  10 Porcinepancreatic ATPDase: Lys Ser Asp Thr Gln Glu Thr Tyr Gly Ala 7                  5                  10 Leu Asp Leu Gly Gly Ala Ser ThrGln Val                  15                  20

[0046] When compared to the sequence which accession number is G2345(CD39 gene product; Maliszenski et al. 1994), the above partialsequences show a very high degree of homology. The following differencesare however found with the CD39 sequence:

[0047] In the porcine pancreatic enzyme, Gln²⁰² is changed to Lys, theAsn²⁰⁴ is changed to Asp, Asn²⁰⁵ is changed to Thr.

[0048] In the bovine aortic enzyme, Arg¹⁴⁷ is changed to Lys, Val¹⁴⁸ ischanged to Ile, Asp¹⁵⁰ is changed to Ala, Gln¹⁵³ is changed to Ala,Arg¹⁵⁴ is changed to Ser, and Leu¹⁵⁶ is changed to Ile.

[0049] The human CD39 has a predicted molecular weight of 57 KDa, whilethe apparent molecular of this protein is 78KDa on SDS-PAGE.

[0050] Both ATPDases type I and II share a high degree of homology withCD39 for the compared sequenced fragments. CD39 appears to be a humanenzyme corresponding to the bovine aortic ATPDase. It is worthwhilenoting that the first N-terminal 200 amino acids of CD39 are absent fromthe ATPDase type I (pancreatic enzyme). This suggests that the activesite of ATPDases is located between the residues 200-510 of CD39 andthat part of CD39 is sufficient to provide this activity. It is furtherworthwhile noting that exact correspondence between the two ATPDases ofthis invention and the already described ATPDases cannot be established.The human placenta ATPDase (Christoforidis et al. 1995) has a molecularweight of 82KDa while CD39 (also of human origin) has a molecular weightof 78KDa. Due to the differences found in diverse tissues of the samespecies, extrapolation cannot be done to the effect that the bovineaorta enzyme of this invention is one of the already described enzymes.The obtained partial amino acid sequences indeed already showndifferences of sequences which may affect some of the physico-chemicalproperties of the claimed enzymes when compared to their humancounterparts (some of the above-observed substitutions are notconservative ones; the net charge of the enzymes may not be the same andthe substituted amino acids may change the behaviour of the enzymes(optimum pH, sensitivity towards inhibitors, etc . . .).

[0051] Cross-reactivity between ATPDases I and II

[0052] Antibodies were produced in rabbits against the following aminoacid sequence which is common to ATPDase I and CD39: SEQ. ID. NO.: LysSer Asp Thr Gln Glu Thr Tyr Gly Ala 8                  5                  10 Leu Asp Leu Gly Gly Ala                 15

[0053]FIG. 5 shows that these antibodies reacted positively with a 78KDaprotein present in endothelial extracts of human sources. They alsoreacted with a protein of 78 KDa of a bovine aorta extract (data notshown). This is an indication that ATPDases I and II share homology ofsequence, and that the latter comprises the peptidic sequence of SEQ.ID. No.: 8 or a variant thereof.

[0054] A type I ATPDase appears to be present in low amounts inendothelial cells as shown by the detection of a faint bandcorresponding to this protein (54KDa) in FIG. 5.

[0055] Conclusions

[0056] Considering that the ATPDase has an antihemostatic role in thesaliva of blood-feeding insects and leeches (Rigbi et al., 1987);

[0057] considering that Côté et al. (1992) have demonstrated bovineATPDase type II has platelet anti-aggregant properties by converting ADPto AMP;

[0058] considering the low Km of the aorta type II enzyme (μM), theoptimum pH of catalysis pH 7.5-8.0, its localization at the surface ofendothelial and smooth muscle cells of blood vessels (Côté et al.,1992);

[0059] considering that the purified enzyme keeps its originalcharacteristics;

[0060] it sounds predictable that the aorta enzyme produced in thepresent invention can be introduced in the circulatory system ofmammalians to reduce platelet aggregation and thrombogenicity.

[0061] Furthermore, considering that a crude microsomal bovine ATPDasetype II fraction has been successfully conjugated to agarose and thatthe conjugate could reduce ADP induced platelet aggregation (Hirota etal., 1987);

[0062] considering that a semi-purified plant ATPDase has beensuccessfully coupled to the following matrices: CM-cellulose, copolymersof L-alanine and L-glutamic acid, polyaspartic acid, polygalacturonicacid, Elvacite 2008™ (methyl methacrylate) and ethylene-maleic acidco-polymer (Patel et al., 1969);

[0063] we propose that the purified ATPDase type II can be coupled toartificial polymers/biomaterials to reduce thrombogenicity (plateletaggregation).

[0064] Therefore, pharmaceutical compositions for use in the reductionof platelet aggregation and thrombogenicity are under the scope of theinvention. These compositions should contain, as an active ingredient,the ATPDase type II of this invention combined to an acceptable carrierwithout excluding any form or formulation of such compositions. Finally,considering that the sequenced CD39 appears to correspond to a humancounterpart of the bovine ATPDase type II enzyme of this invention, theuse of CD39 or variants or a part thereof for reducing plateletaggregation and thrombogenicity is also part of this invention.

[0065] A new process for producing an ATPDase comprising the steps of:

[0066] obtaining a host which comprises a nucleic acid encoding aprotein having the amino acid sequence defined in SEQ. ID. NO.: 1, or avariant thereof, or a part thereof, said variant or part being capableof converting ATP to ADP and ADP to AMP;

[0067] culturing said host in a culture medium supporting the growth ofsaid host and the expression of said nucleic acid;

[0068] recovering the ATP diphosphohydrolase from the culture medium orfrom said host; and

[0069] purifying the ATP diphosphohydrolase is also part of theinvention. Preferably the nucleic acid is the one defined in SEQ ID NO.:2, or a part or a variant thereof, which part or variant is capable ofproducing an ATP diphosphohydrolase.

[0070] The present invention has been described hereinabove; it willbecome apparent to the skilled reader that variations could be broughtthereto without departing from the teachings of the present disclosure.Such variations are under the scope of this invention. TABLE 3 ATPDasepurification Specific Hydrol- activity ysis Total Total (ATP)Purification rates protein activity units/ Yield factor ATP/ Steps mgunits mg % fold ADP ZGM 20.0 60.8 3.0 — (160) *  1.3 ZGM + 20.0 40.6 2.0100 1 1.3 Triton X-100 100,000 g 17.6 37.0 2.1 91   1.1 1.3 supernatantof solubil- ized ZGM DEAE 3.5 28.8 8.3 71   4.2 1.3 column Affi-Gel 0.3113.8 45 34 23  1.3 blue column

[0071] Bibliography

[0072] Baykov et al. (1988). Anal. Biochem. 171:266-270.

[0073] Bradford, M (1976). Anal. Biochem. 72:248-254.

[0074] Côté et al. (1991). BBA 1078:187-191.

[0075] Côté et al. (1992). BBA 1139:133-142.

[0076] Christoforidis et al. (1995), Eur. J. Biochem. 234:66-74.

[0077] Hirota et al. (1987). Thrombosis Res. 45:201-209.

[0078] Laemmli (1970). Nature 227:680-685.

[0079] Lebel et al. (1980). J. Biol. Chem. 255:1227-1233.

[0080] Maliszewki et al. (1994). J. Immunol. :3574-3583

[0081] Patel et al. (1969). BBA 178:626-629.

[0082] Rigbi et al. (1987). Comp. Biochem. Physiol. 87B:567-573.

[0083] Yagi et al. (1989). Eur. J. Biochem. 180:509-513.

1 8 1 510 PRT Homo sapiens 1 Met Glu Asp Thr Lys Glu Ser Asn Val Lys ThrPhe Cys Ser Lys Asn 1 5 10 15 Ile Leu Ala Ile Leu Gly Phe Ser Ser IleIle Ala Val Ile Ala Leu 20 25 30 Leu Ala Val Gly Leu Thr Gln Asn Lys AlaLeu Pro Glu Asn Val Lys 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser SerHis Thr Ser Leu Tyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn AspThr Gly Val Val His Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly ProGly Ile Ser Lys Phe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr LeuThr Asp Cys Met Glu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser GlnHis Gln Glu Thr Pro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met ArgLeu Leu Arg Met Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu AspVal Val Glu Arg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe GlnGly Ala Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly TrpIle Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr ArgTrp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205 PheGly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210 215 220Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230235 240 Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr245 250 255 Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile GlnVal 260 265 270 Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro GlyTyr Lys 275 280 285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro CysThr Lys Arg 290 295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu IleGln Gly Ile Gly 305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile LeuGlu Leu Phe Asn Thr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala PheAsn Gly Ile Phe Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala PheSer Ala Phe Tyr Phe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser GluLys Val Ser Gln Glu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe CysAla Gln Pro Trp Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly ValLys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr IleLeu Ser Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser TrpGlu His Ile His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 GlyTrp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455 460Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr Tyr Val Phe Leu 465 470475 480 Met Val Leu Phe Ser Leu Val Leu Phe Thr Val Ala Ile Ile Gly Leu485 490 495 Leu Ile Phe His Lys Pro Ser Tyr Phe Trp Lys Asp Met Val 500505 510 2 1818 PRT Homo sapiens 2 Ala Cys Cys Ala Cys Ala Cys Cys AlaAla Gly Cys Ala Gly Cys Gly 1 5 10 15 Gly Cys Thr Gly Gly Gly Gly GlyGly Gly Gly Gly Ala Ala Ala Gly 20 25 30 Ala Cys Gly Ala Gly Gly Ala AlaAla Gly Ala Gly Gly Ala Gly Gly 35 40 45 Ala Ala Ala Ala Cys Ala Ala AlaAla Gly Cys Thr Gly Cys Thr Ala 50 55 60 Cys Thr Thr Ala Thr Gly Gly AlaAla Gly Ala Thr Ala Cys Ala Ala 65 70 75 80 Ala Gly Gly Ala Gly Thr CysThr Ala Ala Cys Gly Thr Gly Ala Ala 85 90 95 Gly Ala Cys Ala Thr Thr ThrThr Gly Cys Thr Cys Cys Ala Ala Gly 100 105 110 Ala Ala Thr Ala Thr CysCys Thr Ala Gly Cys Cys Ala Thr Cys Cys 115 120 125 Thr Thr Gly Gly CysThr Thr Cys Thr Cys Cys Thr Cys Thr Ala Thr 130 135 140 Cys Ala Thr AlaGly Cys Thr Gly Thr Gly Ala Thr Ala Gly Cys Thr 145 150 155 160 Thr ThrGly Cys Thr Thr Gly Cys Thr Gly Thr Gly Gly Gly Gly Thr 165 170 175 ThrGly Ala Cys Cys Cys Ala Gly Ala Ala Cys Ala Ala Ala Gly Cys 180 185 190Ala Thr Thr Gly Cys Cys Ala Gly Ala Ala Ala Ala Cys Gly Thr Thr 195 200205 Ala Ala Gly Thr Ala Thr Gly Gly Gly Ala Thr Thr Gly Thr Gly Cys 210215 220 Thr Gly Gly Ala Thr Gly Cys Gly Gly Gly Thr Thr Cys Thr Thr Cys225 230 235 240 Thr Cys Ala Cys Ala Cys Ala Ala Gly Thr Thr Thr Ala ThrAla Cys 245 250 255 Ala Thr Cys Thr Ala Thr Ala Ala Gly Thr Gly Gly CysCys Ala Gly 260 265 270 Cys Ala Gly Ala Ala Ala Ala Gly Gly Ala Gly AlaAla Thr Gly Ala 275 280 285 Cys Ala Cys Ala Gly Gly Cys Gly Thr Gly GlyThr Gly Cys Ala Thr 290 295 300 Cys Ala Ala Gly Thr Ala Gly Ala Ala GlyAla Ala Thr Gly Cys Ala 305 310 315 320 Gly Gly Gly Thr Thr Ala Ala AlaGly Gly Thr Cys Cys Thr Gly Gly 325 330 335 Ala Ala Thr Cys Thr Cys AlaAla Ala Ala Thr Thr Thr Gly Thr Thr 340 345 350 Cys Ala Gly Ala Ala AlaGly Thr Ala Ala Ala Thr Gly Ala Ala Ala 355 360 365 Thr Ala Gly Gly CysAla Thr Thr Thr Ala Cys Cys Thr Gly Ala Cys 370 375 380 Thr Gly Ala ThrThr Gly Cys Ala Thr Gly Gly Ala Ala Ala Gly Ala 385 390 395 400 Gly CysThr Ala Gly Gly Gly Ala Ala Gly Thr Gly Ala Thr Thr Cys 405 410 415 CysAla Ala Gly Gly Thr Cys Cys Cys Ala Gly Cys Ala Cys Cys Ala 420 425 430Ala Gly Ala Gly Ala Cys Ala Cys Cys Cys Gly Thr Thr Thr Ala Cys 435 440445 Cys Thr Gly Gly Gly Ala Gly Cys Cys Ala Cys Gly Gly Cys Ala Gly 450455 460 Gly Cys Ala Thr Gly Cys Gly Gly Thr Thr Gly Cys Thr Cys Ala Gly465 470 475 480 Gly Ala Thr Gly Gly Ala Ala Ala Gly Thr Gly Ala Ala GlyAla Gly 485 490 495 Thr Thr Gly Gly Cys Ala Gly Ala Cys Ala Gly Gly GlyThr Thr Cys 500 505 510 Thr Gly Gly Ala Thr Gly Thr Gly Gly Thr Gly GlyAla Gly Ala Gly 515 520 525 Gly Ala Gly Cys Cys Thr Cys Ala Gly Cys AlaAla Cys Thr Ala Cys 530 535 540 Cys Cys Cys Thr Thr Thr Gly Ala Cys ThrThr Cys Cys Ala Gly Gly 545 550 555 560 Gly Thr Gly Cys Cys Ala Gly GlyAla Thr Cys Ala Thr Thr Ala Cys 565 570 575 Thr Gly Gly Cys Cys Ala AlaGly Ala Gly Gly Ala Ala Gly Gly Thr 580 585 590 Gly Cys Cys Thr Ala ThrGly Gly Cys Thr Gly Gly Ala Thr Thr Ala 595 600 605 Cys Thr Ala Thr CysAla Ala Cys Thr Ala Thr Cys Thr Gly Cys Thr 610 615 620 Gly Gly Gly CysAla Ala Ala Thr Thr Cys Ala Gly Thr Cys Ala Gly 625 630 635 640 Ala AlaAla Ala Cys Ala Ala Gly Gly Thr Gly Gly Thr Thr Cys Ala 645 650 655 GlyCys Ala Thr Ala Gly Thr Cys Cys Cys Ala Thr Ala Thr Gly Ala 660 665 670Ala Ala Cys Cys Ala Ala Thr Ala Ala Thr Cys Ala Gly Gly Ala Ala 675 680685 Ala Cys Cys Thr Thr Thr Gly Gly Ala Gly Cys Thr Thr Thr Gly Gly 690695 700 Ala Cys Cys Thr Thr Gly Gly Gly Gly Gly Ala Gly Cys Cys Thr Cys705 710 715 720 Thr Ala Cys Ala Cys Ala Ala Gly Thr Cys Ala Cys Thr ThrThr Thr 725 730 735 Gly Thr Ala Cys Cys Cys Cys Ala Ala Ala Ala Cys CysAla Gly Ala 740 745 750 Cys Thr Ala Thr Cys Gly Ala Gly Thr Cys Cys CysCys Ala Gly Ala 755 760 765 Thr Ala Ala Thr Gly Cys Thr Cys Thr Gly CysAla Ala Thr Thr Thr 770 775 780 Cys Gly Cys Cys Thr Cys Thr Ala Thr GlyGly Cys Ala Ala Gly Gly 785 790 795 800 Ala Cys Thr Ala Cys Ala Ala ThrGly Thr Cys Thr Ala Cys Ala Cys 805 810 815 Ala Cys Ala Thr Ala Gly CysThr Thr Cys Thr Thr Gly Thr Gly Cys 820 825 830 Thr Ala Thr Gly Gly GlyAla Ala Gly Gly Ala Thr Cys Ala Gly Gly 835 840 845 Cys Ala Cys Thr CysThr Gly Gly Cys Ala Gly Ala Ala Ala Cys Thr 850 855 860 Gly Gly Cys CysAla Ala Gly Gly Ala Cys Ala Thr Thr Cys Ala Gly 865 870 875 880 Gly ThrThr Gly Cys Ala Ala Gly Thr Ala Ala Thr Gly Ala Ala Ala 885 890 895 ThrThr Cys Thr Cys Ala Gly Gly Gly Ala Cys Cys Cys Ala Thr Gly 900 905 910Cys Thr Thr Thr Cys Ala Thr Cys Cys Thr Gly Gly Ala Thr Ala Thr 915 920925 Ala Ala Gly Ala Ala Gly Gly Thr Ala Gly Thr Gly Ala Ala Cys Gly 930935 940 Thr Ala Ala Gly Thr Gly Ala Cys Cys Thr Thr Thr Ala Cys Ala Ala945 950 955 960 Gly Ala Cys Cys Cys Cys Cys Thr Gly Cys Ala Cys Cys AlaAla Gly 965 970 975 Ala Gly Ala Thr Thr Thr Gly Ala Gly Ala Thr Gly AlaCys Thr Cys 980 985 990 Thr Thr Cys Cys Ala Thr Thr Cys Cys Ala Gly CysAla Gly Thr Thr 995 1000 1005 Thr Gly Ala Ala Ala Thr Cys Cys Ala GlyGly Gly Thr Ala Thr Thr 1010 1015 1020 Gly Gly Ala Ala Ala Cys Thr AlaThr Cys Ala Ala Cys Ala Ala Thr 1025 1030 1035 1040 Gly Cys Cys Ala ThrCys Ala Ala Ala Gly Cys Ala Thr Cys Cys Thr 1045 1050 1055 Gly Gly AlaGly Cys Thr Cys Thr Thr Cys Ala Ala Cys Ala Cys Cys 1060 1065 1070 AlaGly Thr Thr Ala Cys Thr Gly Cys Cys Cys Thr Thr Ala Cys Thr 1075 10801085 Cys Cys Cys Ala Gly Thr Gly Thr Gly Cys Cys Thr Thr Cys Ala Ala1090 1095 1100 Thr Gly Gly Gly Ala Thr Thr Thr Thr Cys Thr Thr Gly CysCys Ala 1105 1110 1115 1120 Cys Cys Ala Cys Thr Cys Cys Ala Gly Gly GlyGly Gly Ala Thr Thr 1125 1130 1135 Thr Thr Gly Gly Gly Gly Cys Ala ThrThr Thr Thr Cys Ala Gly Cys 1140 1145 1150 Thr Thr Thr Thr Thr Ala CysThr Thr Thr Gly Thr Gly Ala Thr Gly 1155 1160 1165 Ala Ala Gly Thr ThrThr Thr Thr Ala Ala Ala Cys Thr Thr Gly Ala 1170 1175 1180 Cys Ala ThrCys Ala Gly Ala Gly Ala Ala Ala Gly Thr Cys Thr Cys 1185 1190 1195 1200Thr Cys Ala Gly Gly Ala Ala Ala Ala Gly Gly Thr Gly Ala Cys Thr 12051210 1215 Gly Ala Gly Ala Thr Gly Ala Thr Gly Ala Ala Ala Ala Ala GlyThr 1220 1225 1230 Thr Cys Thr Gly Thr Gly Cys Thr Cys Ala Gly Cys CysThr Thr Gly 1235 1240 1245 Gly Gly Ala Gly Gly Ala Gly Ala Thr Ala AlaAla Ala Ala Cys Ala 1250 1255 1260 Thr Cys Thr Thr Ala Cys Gly Cys ThrGly Gly Ala Gly Thr Ala Ala 1265 1270 1275 1280 Ala Gly Gly Ala Gly AlaAla Gly Thr Ala Cys Cys Thr Gly Ala Gly 1285 1290 1295 Thr Gly Ala AlaThr Ala Cys Thr Gly Cys Thr Thr Thr Thr Cys Thr 1300 1305 1310 Gly GlyThr Ala Cys Cys Thr Ala Cys Ala Thr Thr Cys Thr Cys Thr 1315 1320 1325Cys Cys Cys Thr Cys Cys Thr Thr Cys Thr Gly Cys Ala Ala Gly Gly 13301335 1340 Cys Thr Ala Thr Cys Ala Thr Thr Thr Cys Ala Cys Ala Gly CysThr 1345 1350 1355 1360 Gly Ala Thr Thr Cys Cys Thr Gly Gly Gly Ala GlyCys Ala Cys Ala 1365 1370 1375 Thr Cys Cys Ala Thr Thr Thr Cys Ala ThrThr Gly Gly Cys Ala Ala 1380 1385 1390 Gly Ala Thr Cys Cys Ala Gly GlyGly Cys Ala Gly Cys Gly Ala Cys 1395 1400 1405 Gly Cys Cys Gly Gly CysThr Gly Gly Ala Cys Thr Thr Thr Gly Gly 1410 1415 1420 Gly Cys Thr AlaCys Ala Thr Gly Cys Thr Gly Ala Ala Cys Cys Thr 1425 1430 1435 1440 GlyAla Cys Cys Ala Ala Cys Ala Thr Gly Ala Thr Cys Cys Cys Ala 1445 14501455 Gly Cys Thr Gly Ala Gly Cys Ala Ala Cys Cys Ala Thr Thr Gly Thr1460 1465 1470 Cys Cys Ala Cys Ala Cys Cys Thr Cys Thr Cys Thr Cys CysCys Ala 1475 1480 1485 Cys Thr Cys Cys Ala Cys Cys Thr Ala Thr Gly ThrCys Thr Thr Cys 1490 1495 1500 Cys Thr Cys Ala Thr Gly Gly Thr Thr CysThr Ala Thr Thr Cys Thr 1505 1510 1515 1520 Cys Cys Cys Thr Gly Gly ThrCys Cys Thr Thr Thr Thr Cys Ala Cys 1525 1530 1535 Ala Gly Thr Gly GlyCys Cys Ala Thr Cys Ala Thr Ala Gly Gly Cys 1540 1545 1550 Thr Thr GlyCys Thr Thr Ala Thr Cys Thr Thr Thr Cys Ala Cys Ala 1555 1560 1565 AlaGly Cys Cys Thr Thr Cys Ala Thr Ala Thr Thr Thr Cys Thr Gly 1570 15751580 Gly Ala Ala Ala Gly Ala Thr Ala Thr Gly Gly Thr Ala Thr Ala Gly1585 1590 1595 1600 Cys Ala Ala Ala Ala Gly Cys Ala Gly Cys Thr Gly AlaAla Ala Thr 1605 1610 1615 Ala Thr Gly Cys Thr Gly Gly Cys Thr Gly GlyAla Gly Thr Gly Ala 1620 1625 1630 Gly Gly Ala Ala Ala Ala Ala Ala ThrCys Gly Thr Cys Cys Ala Gly 1635 1640 1645 Gly Gly Ala Gly Cys Ala ThrThr Thr Thr Cys Cys Thr Cys Cys Ala 1650 1655 1660 Thr Cys Gly Cys AlaGly Thr Gly Thr Thr Cys Ala Ala Gly Gly Cys 1665 1670 1675 1680 Cys AlaThr Cys Cys Thr Thr Cys Cys Cys Thr Gly Thr Cys Thr Gly 1685 1690 1695Cys Cys Ala Gly Gly Gly Cys Cys Ala Gly Thr Cys Thr Thr Gly Ala 17001705 1710 Cys Gly Ala Gly Thr Gly Thr Gly Ala Ala Gly Cys Thr Thr CysCys 1715 1720 1725 Thr Thr Gly Gly Cys Thr Thr Thr Thr Ala Cys Thr GlyAla Ala Gly 1730 1735 1740 Cys Cys Thr Thr Thr Cys Thr Thr Thr Thr GlyGly Ala Gly Gly Thr 1745 1750 1755 1760 Ala Thr Thr Cys Ala Ala Thr AlaThr Cys Cys Thr Thr Thr Gly Cys 1765 1770 1775 Cys Thr Cys Ala Ala GlyGly Ala Cys Thr Thr Cys Gly Gly Cys Ala 1780 1785 1790 Gly Ala Thr AlaCys Thr Gly Thr Cys Thr Cys Thr Thr Thr Cys Ala 1795 1800 1805 Thr GlyAla Gly Thr Thr Thr Thr Thr Cys 1810 1815 3 11 PRT Bovine 3 Glu Thr ProVal Tyr Leu Gly Ala Thr Ala Gly 1 5 10 4 5 PRT Bovine 4 Leu Leu Arg MetGlu 1 5 5 13 PRT Bovine UNSURE (8) Xaa, where Xaa = any amino acid 5 AlaAsp Lys Ile Leu Ala Asn Xaa Val Ala Ser Ser Ile 1 5 10 6 10 PRT Bovine 6Tyr Pro Phe Asp Phe Gln Gly Ala Arg Ile 1 5 10 7 19 PRT Porcine 7 LysSer Asp Thr Gln Glu Thr Tyr Gly Ala Leu Asp Leu Gly Gly Ala 1 5 10 15Ser Thr Gln 8 16 PRT Human and bovine 8 Lys Ser Asp Thr Gln Glu Thr TyrGly Ala Leu Asp Leu Gly Gly Ala 1 5 10 15

What is claimed is:
 1. An isolated and purified ATP diphosphohydrolaseobtainable from bovine aorta characterized by the followingphysico-chemical properties: a catalytic unit of a molecular weight ondenaturing polyacrylamide gel electrophoresis of about 78 KDa in itsnative form; a deglycosylated form of said catalytic unit of a molecularweight on SDS-PAGE of about 56 KDa; and characterized in that itcomprises the amino acid sequences defined in SEQ. ID. NOs. 3 to
 6. 2.An ATP diphosphohydrolase as defined in claim 1 further comprising theamino acid sequence defined in SEQ. ID. No.: 8 or a variant thereof. 3.An isolated and purified ATP diphosphohydrolase obtainable from pigpancreatic zymogen granules characterized by the followingphysico-chemical properties: a catalytic unit of a molecular weight ondenaturing polyacrylamide gel electrophoresis of about 54 KDa in itsnative form; a deglycosylated form of said catalytic unit of a molecularweight on SDS-PAGE of about 35 KDa; and characterized in that itcomprises the amino acid sequence defined in SEQ. ID. NO.:
 7. 4. Aprocess for purifying an ATP-diphosphohydrolase enzyme from a tissuecapable to convert ATP to ADP and ADP to AMP which comprises: a)obtaining a sub-cellular microsomal fraction from an homogenate of saidtissue; b) solubilizing said microsomal fraction in the presence of anon-ionic detergent; c) centrifuging said solubilized microsomalfraction to obtain a supernatant containing said enzyme; d) submittingsaid supernatant to an ion-exchange chromatography to obtain a firstenzyme eluate; e) submitting said first eluate to an affinity columnchromatography to obtain a second enzyme eluate; and f) submitting saidsecond eluate to a separation step on a non-denaturing gelelectrophoresis to recover said enzyme free of any contaminant, thepresence of said contaminant being monitored by overstaining said gel ina silver nitrate dye or Coomassie Blue dye.
 5. A process according toclaim 4 wherein said ion exchange chromatography is achieved on a columncontaining Diethylaminoethyl (DEAE).
 6. A process according to claim 5wherein said column is a DEAE agarose column.
 7. A process according toclaim 4 or 5 wherein said affinity column chromatography is achieved onan Affigel™ Blue column.
 8. A process according to claim 4, 5, 6 or 7wherein said non-ionic detergent is Triton X-100™.
 9. A processaccording to claim 4, 5, 6, 7 or 8 wherein an aliquot of said enzyme isfurther submitted after step f) to a polyacrylamide gel electrophoresisunder denaturing conditions to verify its homogeneity and to obtain itsapparent molecular weight.
 10. A process according to claim 9 whereinsaid enzyme is obtained from pig pancreatic zymogen granules and has anapparent molecular weight of 54 Kilodaltons.
 11. A process according toclaim 9 wherein said enzyme is obtained from bovine aortic intima layerand has an apparent molecular weight of about 78 Kilodaltons.
 12. Aprocess according to claim 10 wherein, between steps e) and f), a stepof deglycosylation is included, and whereby the apparent molecularweight is shifted from 54 to 35 KDa.
 13. A process according to claim 11herein, between steps e) and f), a step of deglycosylation is included,and whereby the apparent molecular weight is shifted from 78 to 56 KDa.14. The use of the ATP diphosphohydrolase of claim 1 or 2, for reducingplatelet aggregation and thrombogenicity.
 15. The use of an ATPdiphosphohydrolase for reducing platelet aggregation andthrombogenicity, said ATP diphosphohydrolase having the amino acidsequence defined in SEQ. ID. NO.: 1, or a variant thereof, or a partthereof, said variant or part being capable of converting ATP to ADP andADP to AMP.
 16. A composition for use in the reduction of plateletaggregation and thrombogenicity which comprises as an active ingredientthe ATP diphosphohydrolase of claim 1 or 2 or an ATP diphosphohydrolasewhich sequence is defined in SEQ. ID. NO.: 1, or a variant or a partthereof, which variant or part has an ATP diphosphohydrolase activity,in an acceptable pharmaceutical carrier.
 17. A process for producing anATP diphosphohydrolase which comprises the steps of: obtaining a hostwhich comprises a nucleic acid encoding a protein having the amino acidsequence defined in SEQ. ID. NO.: 1, or a variant thereof, or a partthereof, said variant or part being capable of converting ATP to ADP andADP to AMP; culturing said host in a culture medium supporting thegrowth of said host and the expression of said nucleic acid; recoveringthe ATP diphosphohydrolase from the culture medium or from said host;and purifying the ATP diphosphohydrolase.
 18. A process as defined inclaim 17, wherein said nucleic acid has a sequence defined in SEQ. ID.NO.: 2, a variant thereof or a part thereof, said variant or part beingcapable of producing an ATP diphosphohydrolase which converts ATP to ADPand ADP to AMD.